The invention relates to methods, compositions and kits for reducing stutter in polymerase chain reaction amplification of microsatellites. In certain embodiments, the invention relates to the use of sorbitol in polymerase chain reactions in an amount effective to reduce stutter in the amplification of mononucleotide, dinucleotide, trinucleotide, tetranucleotide and pentanucleotide microsatellites.
Microsatellites, or short tandem repeats (STRs), consist of tandemly repeated DNA sequence motifs of 1 to 6 nucleotides in length. They are widely dispersed and abundant in the eukaryotic genome, and are often highly polymorphic due to variation in the number of repeat units. This polymorphism renders microsatellites attractive DNA markers for genetic mapping, medical diagnostics and forensic investigation. The combination of PCR and gel or capillary electrophoresis under denaturing conditions has greatly improved the genotyping of microsatellite DNA sequences. However, PCR artifacts exhibited by non-proofreading enzymes and referred to as stutter and the terminal transferase side-reaction can complicate analysis of closely spaced microsatellite alleles.
Stutter signals differ from the PCR product representing the genomic allele by multiples of repeat unit size. For dinucleotide repeat loci, the prevalent stutter signal is generally two bases shorter than the genomic allele signal, with additional side-products that are 4 and 6 bases shorter. The multiple signal pattern observed for each allele especially complicates interpretation when two alleles from an individual are close in size (e.g., medical and genetic mapping applications) or when DNA samples contain mixtures from two or more individuals (e.g., forensic applications). Such confusion is maximal for mononucleotide microsatellite genotyping, when both genomic and stutter fragments experience one-nucleotide spacing.
There is a need in the art to develop PCR reaction conditions that minimize or eliminate stutter so that genetic analysis may be more accurate and reliable. This invention is directed to these, as well as other, important ends.
In accordance with some embodiments of the methods of the invention, methods for reducing stutter in the amplification of a microsatellite are provided comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite;
wherein said incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention also provides methods for reducing stutter in the amplification of a mononucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite;
wherein the incubation is performed in the presence of an amount of sorbitol, wherein the sorbitol is effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention also provides methods for reducing stutter in the amplification of a dinucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention further provides methods for reducing stutter in the amplification of a trinucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention further provides methods for reducing stutter in the amplification of a tetranucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention further provides methods for reducing stutter in the amplification of a pentanucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
In further embodiments of the methods of the invention, methods are provided comprising the steps of:
(a) providing a sample comprising a nucleic acid that contains one or more microsatellites selected from the group consisting of mononucleotide microsatellites, dinucleotide microsatellites, trinucleotide microsatellites, tetranucleotide microsatellites and pentanucleotide microsatellites; and
(b) amplifying at least one nucleobase sequence of said nucleic acid, said nucleobase sequence comprising at least one of said microsatellites; said amplified microsatellite having a G+C content of greater than 50%; wherein said amplification is performed in the presence of sorbitol.
Also provided in accordance with the present invention are methods for performing polymerase chain reaction amplification of a microsatellite selected from the group consisting of mononucleotide microsatellites, dinucleotide microsatellites, trinucleotide microsatellites, tetranucleotide microsatellites and pentanucleotide microsatellites, said microsatellite having a G+C content of greater than 50%; said method comprising the step of contacting said microsatellite with a polymerase in the presence of an amount of sorbitol effective to reduce the amount of stutter arising from said amplification relative to the amount of such stutter observed in the absence of sorbitol.
Also provided by the present invention are methods of detecting cancer or a pre-cancerous condition and genetic disorders, in a subject comprising amplifying a region of DNA from a subject, wherein said region comprises a microsatellite selected from the group consisting of a mononucleotide repeat, a dinucleotide repeat, a trinucleotide repeat, a tetranucleotide repeat and a pentanucleotide repeat, wherein said amplification comprises the steps of:
(a) providing a sample comprising a nucleic acid that contains a nucleic acid having a microsatellite instability,
(b) amplifying at least one nucleobase sequence of the nucleic acid, in which the nucleobase sequence comprises at least one of the microsatellites; and
(c) detecting alterations of the microsatellite as compared to corresponding microsatellites amplified from control tissue; the amplified microsatellite having a G+C content of greater than 50%; wherein the amplification is performed in the presence of a sufficient amount of sorbitol effective to reduce stutter from that observed in the absence of sorbitol.
In some embodiments, the cancer or cancerous condition is chronic lymphocytic leukemia. In further embodiments, the microsatellite amplification comprises at least one genetic locus, for example, CAG/CTG, CCG/CGG, and CGA/TCG.
In some embodiments, the genetic disorder is Huntington""s disease or a spinocerebellar ataxia. In further embodiments, the microsatellite amplification comprises at least one genetic locus, for example, CAG/CTG.
In other embodiments, the disorder is a psychiatric disorder. In further embodiments, the microsatellite amplification comprises at least one genetic locus, for example, CCCCT/AGGGG.
In some embodiments, the present invention also provides methods of genetic mapping comprising amplifying a plurality of regions of DNA from a sample containing DNA from a subject, wherein the regions comprise at least one microsatellite selected from the group consisting of a mononucleotide repeat, a dinucleotide repeat, a trinucleotide repeat, a tetranucleotide repeat and a pentanucleotide repeat, wherein the amplification comprises the steps of:
(a) contacting said DNA with a enzyme at least one enzyme having nucleic acid polymerase activity; and
(b) incubating said sample with the enzyme for a time and under conditions sufficient to amplify the regions; and
(c) separating amplified regions, forming a microsatellite pattern; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
In further embodiments, the present invention also provides methods of personal genetic identification comprising amplifying a plurality of regions of DNA from a sample containing DNA from a subject, wherein said regions comprise at least one microsatellite selected from the group consisting of a mononucleotide repeat, a dinucleotide repeat, a trinucleotide repeat, a tetranucleotide repeat and a pentanucleotide repeat; wherein the microsatellite has a G+C content of greater than 50%; wherein the amplification comprises the steps of:
(a) contacting said DNA with a enzyme at least one enzyme having nucleic acid polymerase activity; and
(b) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the regions;
(c) separating amplified regions, forming a microsatellite pattern; and
(d) comparing the microsatellite pattern with a corresponding microsatellite pattern derived from the a DNA sample from a second source; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
In some embodiments, the subject is a forensic sample and a said second source comprises at least one selected from the group consisting of the presumed matching source, a family member of the presumed matching source, and a database of sources.
In some embodiments of the methods of the invention, where the microsatellite is a dinucleotide microsatellite, the microsatellite comprises a dinucleotide repeat is CG/CG. In further embodiments where the microsatellite is a trinucleotide microsatellite, the microsatellite comprises a trinucleotide repeat selected from the group consisting of CAG/CTG, CCG/CGG, and CGA/TCG. In further embodiments where the microsatellite is a tetranucleotide microsatellite, the microsatellite comprises a tetranucleotide repeat consisting of TGCC/GGCA. In further embodiments where the microsatellite is a pentanucleotide microsatellite, the microsatellite comprises a pentanucleotide repeat consisting of CCCCT/AGGGG.
In some embodiments of the method of the invention, the microsatellite has a G+C content of greater than 50%. In further embodiments, the microsatellite has a G+C content of greater than 66%. In further embodiments, the microsatellite has a G+C content of 75% or more. In further embodiments, the microsatellite has a G+C content of 100%.
In some embodiments of the invention, the amount of stutter is reduced to 90% or less than the amount of stutter obtained in the absence of sorbitol. In other embodiments the amount of stutter is reduced to 80% or less. In other embodiments, the amount of stutter is reduced to 70% or less. In other embodiments, the amount of stutter is reduced to 60% or less. In other embodiments, the amount of stutter is reduced to 50% or less. In other embodiments, the amount of stutter is reduced to 40% or less. In other embodiments, the amount of stutter is reduced to 30% or less.
In some embodiments of the methods of the invention, the amplification comprises contacting said nucleobase sequence with an enzyme having a polymerase activity. For example, the enzyme having polymerase activity may be selected from the group consisting of a DNA polymerase from Thermus aquaticus, Thermus thermophilus, other Thermus species, Bacillus species, Thermococcus species, Thermotoga species, and Pyrococcus species. For example, suitable polymerases include AmpliTaq Gold(copyright) DNA polymerase; AmpliTaq(copyright) DNA Polymerase; AmpliTaq(copyright) DNA Polymerase, Stoffel fragment; rTth DNA Polymerase; rTth DNA Polymerase XL; Bst DNA polymerase large fragment from Bacillus stearothermophilus; Vent and Vent Exo- from Thermococcus litoralis; Tma from Thermotoga maritima; Deep Vent and Deep Vent Exo- and Pfu from Pyrococcus; and mutants, variants and derivatives thereof.
Also provided in certain embodiments of the invention are compositions comprising:
(a) a nucleic acid sequence comprising a microsatellite, in which the microsatellite has a G+C content of greater than 50%, the microsatellite being selected from the group consisting of mononucleotide microsatellites, dinucleotide microsatellites, trinucleotide microsatellites, tetranucleotide microsatellites and pentanucleotide microsatellites;
(b) at least two primers, each of said primers having a sequence that is substantially complementary to a portion of the nucleic acid sequence that is adjacent to the microsatellite;
(c) at least one enzyme having nucleic acid polymerase activity; and (d) sorbitol.
In some embodiments of the methods and compositions of the invention, sorbitol is present in an amount of from 1.5 to 3.5 M. In other embodiments, sorbitol is present in an amount of 2.0 to 3.0 M. In other embodiments, sorbitol is present in an amount of 2.0 M.
In some embodiments of the invention, at least 0.5 mM each of dNTPs are used. In other embodiments, at least 1 mM dNTPs are used.
In some embodiments, the present invention also provides kits for amplification of a target nucleic acid sequence, the target nucleic acid sequence comprising a microsatellite having a G+C content of greater than 50%, selected from the group consisting of mononucleotide microsatellites, dinucleotide microsatellites, trinucleotide microsatellites, tetranucleotide microsatellites, and pentanucleotide microsatellites comprising, in separate containers: a polymerase, a plurality of deoxynucleotide triphosphates; and sorbitol. In some embodiments of the compositions and kits of the invention, the polymerase is selected from the group consisting of a DNA polymerase from Thermus aquaticus, Thermus thermophilus, other Thermus species, Bacillus species, Thermococcus species, Thermotoga species, and Pyrococcus species. For example, suitable polymerases include, but are not limited to, AmpliTaq Gold(copyright) DNA polymerase; AmpliTaq(copyright) DNA Polymerase; AmpliTaq(copyright) DNA Polymerase, Stoffel fragment; rTth DNA Polymerase; rTth DNA Polymerase XL; Bst DNA polymerase large fragment from Bacillus stearothermophilus; Vent and Vent Exo- from Thermococcus litoralis; Tma from Thermotoga maritima; Deep Vent and Deep Vent Exo- and Pfu from Pyrococcus; and mutants, variants and derivatives thereof.
In some further embodiments of the invention, methods are provided in which a sample containing nucleic acid that is suspected of containing one or more microsatellites having a G+C content of greater than 50% is contacted with an enzyme that polymerizes nucleotides in the presence of an effective amount of sorbitol to reduce observed stutter relative to the amount of stutter observed in the absence of sorbitol, and amplifying at least one nucleobase sequence containing at least one microsatellite of the nucleic acid contained in the sample. Such microsatellites may include mononucleotide, dinucleotide, trinucleotide, tetranucleotide microsatellites and/or pentanucleotide microsatellites.